Articles pubs.acs.org/acschemicalbiology
Deciphering Structural Elements of Mucin Glycoprotein Recognition Andrew Borgert,†,# Jamie Heimburg-Molinaro,‡,# Xuezheng Song,‡ Yi Lasanajak,‡ Tongzhong Ju,‡ Mian Liu,§ Pamela Thompson,§ Govind Ragupathi,∥ George Barany,⊥ David F. Smith,‡ Richard D. Cummings,*,‡ and David Live*,§ †
Center for Magnetic Resonance Research and ⊥Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States ‡ Department of Biochemistry, Emory University, Atlanta, Georgia 30322, United States § Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, United States ∥ Memorial Sloan-Kettering Cancer Center, New York, New York 10065, United States S Supporting Information *
ABSTRACT: Mucin glycoproteins present a complex structural landscape arising from the multiplicity of glycosylation patterns afforded by their numerous serine and threonine glycosylation sites, often in clusters, and with variations in respective glycans. To explore the structural complexities in such glycoconjugates, we used NMR to systematically analyze the conformational effects of glycosylation density within a cluster of sites. This allows correlation with molecular recognition through analysis of interactions between these and other glycopeptides, with antibodies, lectins, and sera, using a glycopeptide microarray. Selective antibody interactions with discrete conformational elements, reflecting aspects of the peptide and disposition of GalNAc residues, are observed. Our results help bridge the gap between conformational properties and molecular recognition of these molecules, with implications for their physiological roles. Features of the native mucin motifs impact their relative immunogenicity and are accurately encoded in the antibody binding site, with the conformational integrity being preserved in isolated glycopeptides, as reflected in the antibody binding profile to array components.
T
N-acetylgalactosaminyltransferases (ppGalNAcTs),4 followed by elaboration with other sugars to generate complex O-glycans. The glycosylation patterns generated by these enzymes are conferred by the catalytic domains5 and, if proximal O-GalNAcs are already in place, influenced by their lectin domains.6,7 However, the lack of strictly defined consensus sequences for O-glycosylation,5 together with the known heterogeneity in O-glycan structures, creates challenges for defining discrete mucin recognition elements. Characterization of O-glycosylation by mass spectral methods typically relies on chemically released glycans, with loss of crucial sequence-specific data on sites of modification. Non-destructive analysis with lectins of known carbohydrate epitope preferences is also commonly used,8 but lectins are largely insensitive to the glycoconjugate context. Their apparent affinities may reflect the degree to which pendant glycans are clustered, providing some basis for selectivity, but this can be rationalized in global thermodynamic terms without a detailed structural knowledge.9 These analytical limitations have given rise to an intrinsic ambiguity in defining the O-glycan epitopes. For instance, serine/threonine α-O-GalNAc is referred to as the
he number of glycan structures of the human glycome is estimated to be many thousands.1 This diversity, arising from the variety of residues and multiple linkage options, is also amplified by glycan conjugation to other components including proteins and lipids. A large fraction of mammalian proteins are glycosylated,2 and the combinatorial possibilities of these glycoconjugates present a complexity unparalleled in genomics and proteomics, further compounded by the intrinsic heterogeneity in glycoproteins, thought to be a consequence of nontemplate driven glycosylation. Additionally, cellular regulation of glycan structures and patterns through differential enzyme expression in the normal or disease states allows cell surface glycoproteins to function as temporally regulated biomarkers. The endogenous presentation of aberrant glycosylation can also give rise to circulating antibodies that serve as secondary markers.3 Given their prominence in communication between cells and surroundings, understanding the contributions made by the protein and glycan components to molecular recognition are critical in how information is encoded for specific glycoprotein interactions and functions. Mucin-type O-glycosylation, characterized by a prevalence of threonine and serine residues modified with N-acetylgalactosamine (GalNAc), constitutes a major and complex form of protein modification, encountered on the cell surface. Mucin O-glycan biosynthesis occurs in a stepwise fashion, initiated by members of a family of about two dozen polypeptide © 2012 American Chemical Society
Received: December 15, 2011 Accepted: March 23, 2012 Published: March 23, 2012 1031
dx.doi.org/10.1021/cb300076s | ACS Chem. Biol. 2012, 7, 1031−1039
ACS Chemical Biology
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Tn antigen, not discriminating the amino acid to which it is attached,10 providing insufficient definition of this epitope in the context of a glycoprotein. This structure, which is normally rare in humans, is relevant because of the correlation of its aberrant appearance with poor prognosis in cancer, where altered densities and clustering are observed.11−13 It has been a target in diagnostic and therapeutic strategies, particularly in development of glycoconjugate antitumor vaccines.12,14 Understanding the conformation of mucins even with the minimal Tn antigen is broadly relevant to mucin structural biology since the α-O-GalNAc residue of their glycans is key in organizing the core glycoprotein scaffold15 underlying potentially more complex pendant glycans. The relevance of more accurate characterization of epitopes with S/T-α-O-GalNAc is indicated by the differential recognition of glycosylation in isolated sites or in clusters by antibodies in normal immune responses and those induced in therapeutic applications of glycoconjugate vaccines.3,12,16 Results from surface plasmon resonance17 and array binding studies3,18,19 show that antibody recognition of mucin structures is influenced by presentation in the glycoconjugate environment, but these findings have not been accompanied by any structural studies. The importance of this is illustrated in a recent crystal structure of a T-α-O-GalNAc glycopeptide−antibody complex showing contacts between the antibody and both carbohydrate and peptide portions.20 Since material isolated from natural sources displays microheterogeneity even if isolated from a single cell type, we have employed chemical synthesis to provide homogeneous well-defined material needed for biophysical studies using NMR methods for a systematic analysis of mucin conformation as a function of glycosylation density. The constructs examined in this way and others have been assembled in a glycopeptide microarray to gain further insight into how conformational properties mediate binding of lectins and antibodies.
Table 1. Peptide Backbone and Threonine Side Chain Vicinal Coupling Constants (Hz)a construct PEP
A
B
C
D
E
F
G
8.6 8.3 8.0 5.7 6.7
7.8 8.9 8.5 5.5 7.3
8.9 9.5 8.5 5.5 7.0
2.6 4.2
